Throughout this application various publications are referred to by partial citations within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Neuroregulators comprise a diverse group of natural products that subverse or modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids, and lipid metabolites, and other metabolic byproducts. Many of these neuroregulator substances interact with specific cell surface receptors, which transduce signals from the outside to the inside of the cell. G-protein coupled receptors (GPCRs) represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are characterized by seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequelae such as stimulation of adenylyl cyclase.
Vitamin A1 (all-trans-retinol) is oxidized to vitamin A1 aldehyde (all-trans-retinal) by an alcohol dehydrogenase. All-trans-retinal is critical for the synthesis of rhodopsin in retinal cells, where it plays a key role in the visual system. All-trans-retinal can also be converted to all-trans-retinoic acid (ATRA) by aldehyde dehydrogenase and oxidase in other cell types (Bowman, W. C. and Rand, M. J., 1980).
Historically, ATRA and the other active metabolites of vitamin A, 9-cis-retinoic acid (9CRA), were thought to only mediate their cellular effects through the action of nuclear retinoic acid receptors (RARxcex1, xcex2, xcex3) and retinoid X receptors (RXRxcex1, xcex2, xcex3) (Mangelsdorf, D. J., et al, 1994) These receptors are members of a superfamily of ligand-dependent transcription factors, which include the vitamin D receptor (VDR), thyroid hormone receptor (TR), and peroxisome proliferator activator receptors (PPAR). They form heterodimers and homodimers that bind to DNA response elements in the absence of ligand. In response to ligand binding the dimer changes conformation which leads to transactivation and regulation of transcription of a set(s) of cell type-specific genes (Mangelsdorf, D. J., et al, 1994; Hofman, C. and Eichele, G., 1994; and Gudas, L. J. et al, 1994).
Since retinoic acid produces a wide variety of biological effects, it is not surprising that it is proposed to play an important role in various physiological and pathophysiological processes. Retinoids control critical physiological events including cell growth, differentiation, reproduction, metabolism, and hematopoiesis in a wide variety of tissues. At a cellular level, retinoids are capable of inhibiting cell proliferation, inducing differentiation, and inducing apoptosis (Breitman, T. et al, 1980; Sporn, M. and Roberts, A., 1984, and Martin, S., et al, 1990). These diverse effects of retinoid treatment prompted a series of investigations evaluating retinoids for cancer chemotherapy as well as cancer chemoprevention. Clinically, retinoids are used for the treatment of a wide variety of malignant diseases including: acute promyelocytic leukemia (APL), cutaneous T-cell malignancies, dermatological malignancies, squamous cell carcinomas of skin and of the cervix and neuroblastomas (Redfern, C. P. et al, 1995 for review). Retinoids have also been examined for their ability to suppress carcinogenesis and prevent development of invasive cancer. 13-cis retinoic acid reverses oral leukoplakia, the most common premalignant lesion of the aerodigestive tract, and is also used in the chemoprevention of bladder cancer (Sabichi, A. L. et al, 1998, for review). Also, 13-cis retinoic acid treatment as adjuvant therapy after surgery and radiation in head and neck cancer caused a significant delay in the occurrence of second primary cancers (Gottardis, M. M. et al, 1996, for review).
Interestingly, retinoids also have an effect on pancreatic function. It has been demonstrated that retinoic acid (or retinol) is required for insulin secretion from isolated islets (Chertow, B. S., et al, 1987) and from RINm5F rat insulinoma cells (Chertow, B. S., et al, 1989). Retinoic acid may also have an effect on cell-to-cell adhesion and aggregation (Chertow, B. S., et al, 1983). In addition, a single intragastric administration of 9CRA (but not ATRA) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys (Ohmura, T., et al, 1997). Therefore, retinoic acid could play a role in the normal pancreatic function and possibly in the development of diabetes. There is also some evidence that retinoids could be useful in the treatment of pancreatic malignancies (El-Metwally, T. H. et al, 1999; Rosenwicz, S. et al, 1997; and Rosenwicz, S. et al, 1995).
Retinoids have been shown to affect epidermal cell growth and differentiation as well as sebaceous gland activity and exhibit immunomodulatory and anti-inflammatory properties. Therefore, retinoids have been increasingly used for treatment of a variety of skin disorders including: psoriasis and other hyperkeratotic and parakeratotic skin disorders, keratotic genodermatosis, severe acne and acne-related dermatoses, and also for therapy and/or chemoprevention of skin cancer and other neoplasia (Orfanos, C. E., et al, 1997 for review).
Retinoids are also involved in lung development. Fetal lung branching leading to development of the alveolar tree is accelerated by retinoic acid. Currently, prematurely delivered infants who have immature lungs are treated with vitamin A, but other applications may exist that require further investigation (Chytil, F., 1996).
Lastly, there is some evidence that suggests that retinoids may play a role in schizophrenia (Goodman, A. B. 1998) and Alzheimer""s disease (Connor, M. J. and Sidell, N., 1997).
The extensive list of retinoid-mediated effects indicate that retinoic acid receptors (non-nuclear) are attractive as targets for therapeutic intervention for several disorders and would be useful in developing drugs with higher specificity and fewer side effects for a wide variety of diseases.
Platelet-Activating Factor (PAF) is a lipid mediator with multitude of physiological and pathophysiological effects. Originally recognized as a xe2x80x98soluble factorxe2x80x99 responsible for serotonin secretion (Henson, 1970), its chemical identity was revealed in 1979 when Demopoulos et al. demonstrated that a semisynthetic phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine had properties identical to PAF. Naturally-occurring PAF is in fact a mixture of phospholipids containing the alkyl side chains of varying lengths. The exact composition of naturally-occuring PAF is dependent on the site of biosynthesis. A wide variety of cells, such as leukocytes, neutrophils, endothelial cells, platelets and macrophages, can synthesize PAF (Chao and Olson, 1993).
PAF can be generated via two pathways: de novo and remodeling pathways (Maclennan et al., 1996). The precursor in the de novo pathway is 1-alkyl-2-lyso-sn-glycero-3-phosphate which is, several enzymatic steps later, converted to PAF. Alternatively, in the remodeling pathway, PAF is synthesized from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine via the actions of the enzymes phospholipase A2 (PLA2) and acetyltransferase. The intermediates in this pathway are free polyunsaturated fatty acid, such as arachidonic acid, and lyso-PAF. A critical difference between the de novo and remodeling pathways is that, while the former pathway may be responsible for physiological levels of PAF, the latter pathway is believed to be activated only upon stimulation of cells leading to abnormally high levels of PAF. Some of the potent stimuli for PAF secretion involve thrombin, bradykinin and tumor necrosis factor. Additionally, PAF itself can enhance its own synthesis and secretion.
PAF binding has been observed in numerous cell types, suggesting that specific PAF receptors exist in different cells (Chao and Olson, 1993). Platelets from several species exhibit high affinity binding sites for [3H]-PAF with the Kd values ranging from 0.5 nM to 37 nM. In contrast, rat platelets show only nonspecific binding to [3H]-PAF, perhaps explaining the lack of platelet aggregating response to PAF in these cells (Sanchez-Crespo et al., 1981). Specific binding sites for [3H]-PAF are also present on smooth muscle cells, leukocytes, macrophages, and Kupffer cells (Chao et al., 1989; Hwang et al., 1983; Ng and Wong, 1988; Valone, 1988). The [3H]-PAF binding on human neutrophils reveals two binding sites, a high affinity site with the Kd value of 0.2 nM and a low affinity site with the Kd value of 500 nM (Chao and Olson, 1993). [3H]-PAF binding is observed also in the CNS-associated cells such as NG108-15 cells as well as in the CNS areas such as hypothalamus and cerebral cortex (Chau et al., 1992; Hosford et al., 1990; Junier et al., 1988; Marcheselli et al., 1990). Interestingly, high affinity sites on rat cerebral cortex correspond to intracellular sites on microsomal membranes while the low affinity site is present on the plasma membrane (Marcheselli et al., 1990).
PAF produces a diverse array of intracellular actions. Several cell types release arachidonic acid in response to PAF stimulation. This action of PAF may involve the heterotrimeric G-protein and PLA2 since it is blocked by pertussis toxin and the PLA2 inhibitors (Nakashima et al., 1989). Importantly, some physiological actions of PAF are mediated through arachidonic acid metabolites such as leukotrienes and prostaglandins. For example, PAF induced coronary vasoconstriction in the isolated perfused rat heart through the release of LTC4 (Piper and Stewart, 1986). Similarly, PAF-mediated pulmonary vasoconstriction and edema in isolated lungs were accompanied by increased LTC4 and LTD4 levels in the lung effluent perfusate (Voelkel et al., 1982). In addition to arachidonic release, PAF also induces phosphoinositide turnover and increase in intracellular calcium in a wide variety of cells such as platelets, neutrophils and macrophages (Chao and Olson, 1993). Hydrolysis of phosphatidylinositol 4,5-bisphosphate may involve a specific phospholipase C, leading to formation of two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. Prpic et al. (1988) demonstrated that PAF-induced increase in intracellular calcium in macrophages was the result of inositol phosphate generation. However, in rabbit platelets, PAF induces elevation of intracellular calcium via influx of extracellular calcium through calcium channels since the calcium channel blocker could block the PAF action (Lee et al., 1981; Lee et al., 1983). Other biochemical effects of PAF include stimulation of tyrosine phosphorylation of cellular proteins and induction of immediate early genes such as c-fos and c-jun (Chao and Olson, 1993).
Several studies have linked the PAF binding and signaling to the heterotrimeric G-proteins. PAF stimulated GTPase activity in platelets, and GTP caused a shift in PAF binding (Shukla, 1992). Additionally, injection of an inactive GTP analogue reduced PAF-induced chloride current in oocytes (Shimizu et al., 1992). These evidences suggested that PAF might produce its cellular effects via a GPCR. This was confirmed upon cloning of a PAF receptor from guinea pig lungs in 1991 (Honda et al., 1991). This receptor and its species homologues are predicted to have seven transmembrane-spanning regions and contain several highly conserved amino acids present in other GPCRs (Bito et al., 1994; Honda et al., 1991; Nakamura et al., 1991). In a heterologous expression system, the PAF receptor activates primarily the Gq family of G-proteins, although the stimulation of Gi class of G-proteins has also been suggested (Honda et al., 1994).
Through the cloned PAF receptor and possibly through as yet unidentified other PAF receptors, PAF produces a diverse array of physiological and pathophysiological effects. As the name suggests, PAF is a potent activator of platelet aggregation in many species, the notable exception being rat platelets, and enhances secretion of thromboxanes from platelets (Chao and Olson, 1993). It also stimulates aggregation of monocytes and leukocytes (Ford-Hutchinson, 1983; Yasaka et al., 1982), synthesis of leukotrienes from leukocytes (Gorman et al., 1983), and degranulation of eosinophils (Bartemes et al., 1999). In addition, it behaves as a chemotactic factor for several cell types such as monocytes and eosinophils (Del Sorbo et al., 1999; Liu et al., 1998). When injected intravenously, it can cause thrombocytopenia and leukopenia (Demopoulos et al., 1981).
PAF has potent effects on cardiovascular parameters. When given systemically, it causes vasodilation and hypotension (Handa et al., 1990; Yamanaka et al., 1992). However, its effect on coronary and pulmonary circulations is dependent on the dose (Goldstein et al., 1986). It also increases vascular permeability, allowing plasma extravasation. When infused into the carotid artery, PAF causes a decrease in cerebral blood flow (Kochanek et al., 1988). Similarly, PAF administration reduces spinal cord blood flow (Faden and Holt, 1992). These effects of PAF are independent of any direct action on cerebral vasculature, but may be the result of the change in blood-brain barrier (Kumar et al., 1988). PAF contracts other smooth muscles such as gastrointestinal, uterine and pulmonary smooth muscles directly as well as via release of other mediators (Martinez-Cuesta et al., 1996; Pritze et al., 1991; Zhu et al., 1992). It also contracts the airway smooth muscle, increasing airway resistance and responsiveness to other bronchoconstrictors (Austin and Foreman, 1993; Nagase et al., 1997). This mechanism may contribute to the role of PAF in asthma.
In addition to several effects in periphery, PAF plays an important role in various CNS-associated processes (Maclennan et al., 1996). It has been suggested that PAF is involved in long-term potentiation (LTP) (Bazan, 1998). Application of PAF antagonist inhibited the development of LTP, and PAF induction of LTP prevented subsequent high-frequency stimulation-induced LTP (Del Cerro et al., 1990; Wierraszko et al., 1993). Furthermore, PAF increases glutamate release in the brain, a property expected in a retrograde messenger involved in LTP (Kato et al., 1994). Administration of PAF also modulates levels of adrenocorticotrophic hormone, beta-endorphin and corticosterone (Maclennan et al., 1996). Furthermore, PAF can regulate neuronal differentiation in cultured rat cerebral neurons and NG 108-15 cells (Kornecki and Ehrlich, 1988; Ved et al., 1991).
Due to its varied biological properties, PAF has been suggested to play a pivotal role in many pathophysiological processes, for example, inflammation and allergy, and ischemia-reperfusion injury (Maclennan et al., 1996). PAF acts directly on leukocytes as well as promotes interaction of leukocytes and endothelial cells, leading to the activation of leukocytes and release of inflammatory mediators, such as oxygen radicals, cytokines, prostaglandins and leukotrienes. Some of the inflammatory diseases that may involve PAF as a mediator are acute and chronic pancreatitis, acute renal failure, chronic bowel inflammation, Crohn""s disease, ulcerative colitis, rheumatoid arthritis, psoriasis, sepsis and septic shock, cutaneous inflammation, bacterial meningitis, inflammatory bullous diseases and acute endotoxemia (Fink, 1998; Gardner et al., 1995; Heller et al., 1998; Johnson, 1999; Konturek et al., 1992; Lopez-Novoa 1999; Loucks et al., 1997; Stack and Hawkey, 1997; Zhou et al., 1990). PAF also produces pathological features characteristic of asthma (Heller et al., 1998; Page, 1992). It constricts bronchial tissue, produces tracheal and bronchial edema, stimulates secretion of mucus and causes bronchial hyperresponsiveness. PAF also produces many signs and symptoms of anaphylactic shock, suggesting its potential role in this condition (Lefort et al., 1992). Furthermore, PAF may play an important role in ischemia-reperfusion injury in various tissues such as heart and brain (Loucks et al., 1997; Maclennan et al., 1996). And finally, it has been suggested that PAF is an HIV-1-induced neurotoxin and plays a role in HIV-associated dementia (Maclennen et al., 1996).
In summary, PAF is a potent lipid mediator with a variety of biological actions. It is suggested to play a pivotal role in various pathophysiological conditions. Therefore, any ligand targeted towards altering PAF synthesis, biological actions and degradation may provide useful pharmacological therapies.
This invention provides an isolated nucleic acid encoding a mammalian SNORF25 receptor.
This invention further provides a purified mammalian SNORF25 receptor protein.
This invention also provides a vector comprising a nucleic acid in accordance with this invention.
This invention still further provides a cell comprising a vector in accordance with this invention.
This invention additionally provides a membrane preparation isolated from a cell in accordance with this invention.
Furthermore, this invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF25 receptor, wherein the probe has a sequence complementary to a unique sequence present within one of the two strands of the nucleic acid encoding the mammalian SNORF25 receptor contained in plasmid pEXJT3T7-hSNORF25 (ATCC Accession No. 203495).
This invention further provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian SNORF25 receptor, wherein the probe has a sequence complementary to a unique sequence present within (a) the nucleic acid sequence shown in FIGS. 1A-1B (SEQ ID NO. 1) or (b) the reverse complement thereof.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to RNA encoding a mammalian SNORF25 receptor, so as to prevent translation-of such RNA.
This invention further provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to genomic DNA encoding a mammalian SNORF25 receptor, so as to prevent transcription of such genomic DNA.
This invention also provides an antibody capable of binding to a mammalian SNORF25 receptor encoded by a nucleic acid in accordance with this invention.
Moreover, this invention provides an agent capable of competitively inhibiting the binding of an antibody in accordance with this invention to a mammalian SNORF25 receptor.
This invention still further provides a pharmaceutical composition comprising (a) an amount of an oligonucleotide in accordance with this invention capable of passing through a cell membrane and effective to reduce expression of a mammalian SNORF25 receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.
This invention also provides a pharmaceutical composition which comprises an amount of an antibody in accordance with this invention effective to block binding of a ligand to a human SNORF25 receptor and a pharmaceutically acceptable carrier.
This invention further provides a transgenic, nonhuman mammal expressing DNA encoding a mammalian SNORF25 receptor in accordance with this invention.
This invention still further provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of a native mammalian SNORF25 receptor.
This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a mammalian SNORF25 receptor in accordance with this invention so placed within such genome as to be transcribed into antisense mRNA which is complementary to and hybridizes with mRNA encoding the mammalian SNORF25 receptor so as to reduce translation of such mRNA and expression of such receptor.
This invention provides a process for identifying a chemical compound which specifically binds to a mammalian SNORF25 receptor which comprises contacting cells containing DNA encoding, and expressing on their cell surface, the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF25 receptor.
This invention further provides a process for identifying a chemical compound which specifically binds to a mammalian SNORF25 receptor which comprises contacting a membrane preparation from cells containing DNA encoding, and expressing on their cell surface, the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian SNORF25 receptor.
This invention still further provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF25 receptor which comprises separately contacting cells expressing on their cell surface the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chemical compound to the mammalian SNORF25 receptor, a decrease in the binding of the second chemical compound to the mammalian SNORF25 receptor in the presence of the chemical compound being tested indicating that such chemical compound binds to the mammalian SNORF25 receptor.
This invention further provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian SNORF25 receptor which comprises separately contacting a membrane preparation from cells expressing on their cell surface the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of such compounds to the receptor, and detecting specific binding of the chemical compound to the mammalian SNORF25 receptor, a decrease in the binding of the second chemical compound to the mammalian SNORF25 receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian SNORF25 receptor.
In an embodiment of the invention, the second compound is a lipid-like molecule including, but not limited to, ATRA and phospholipids. Examples of phospholipids include, but are not limited to, PAF(C18), PAF(C16), lyso-PAF(C18) and lyso-PAF(C16).
This invention further provides a compound identified by one of the processes of this invention.
This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF25 receptor to identify a compound which specifically binds to the mammalian SNORF25 receptor, which comprises (a)contacting cells transfected with, and expressing, DNA encoding the mammalian SNORF25 receptor with a compound known to bind specifically to the mammalian SNORF25 receptor; (b)contacting the cells of step (a) with the plurality of compounds not known to bind specifically to the mammalian SNORF25 receptor, under conditions permitting binding of compounds known to bind to the mammalian SNORF25 receptor; (c) determining whether the binding of the compound known to bind to the mammalian SNORF25 receptor is reduced in the presence of the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian SNORF25 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the mammalian SNORF25 receptor.
This invention further provides a method of screening a plurality of chemical compounds not known to bind to a mammalian SNORF25 receptor to identify a compound which specifically binds to the mammalian SNORF25 receptor, which comprises (a) contacting a membrane preparation from cells transfected with, and expressing, DNA encoding the mammalian SNORF25 receptor with the plurality of compounds not known to bind specifically to the mammalian SNORF25 receptor under conditions permitting binding of compounds known to bind to the mammalian SNORF25 receptor; (b) determining whether the binding of a compound known to bind to the mammalian SNORF25 receptor is reduced in the presence of any compound within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (c) separately determining the binding to the mammalian SNORF25 receptor of each compound included in the plurality of compounds, so as to thereby identify any compound included therein which specifically binds to the mammalian SNORF25 receptor.
This invention also provides a method of detecting expression of a mammalian SNORF25 receptor by detecting the presence of mRNA coding for the mammalian SNORF25 receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe according to this invention under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the mammalian SNORF25 receptor by the cell.
This invention further provides a method of detecting the presence of a mammalian SNORF25 receptor on the surface of a cell which comprises contacting the cell with an antibody according to this invention under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of the mammalian SNORF25 receptor on the surface of the cell.
This invention still further provides a method of determining the physiological effects of varying levels of activity of mammalian SNORF25 receptors which comprises producing a transgenic, nonhuman mammal in accordance with this invention whose levels of mammalian SNORF25 receptor activity are varied by use of an inducible promoter which regulates mammalian SNORF25 receptor expression.
This invention additionally provides a method of determining the physiological effects of varying levels of activity of mammalian SNORF25 receptors which comprises producing a panel of transgenic, nonhuman mammals in accordance with this invention each expressing a different amount of mammalian SNORF25 receptor.
Moreover, this invention provides a method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF25 receptor comprising administering a compound to a transgenic, nonhuman mammal according to this invention, and determining whether the compound alleviates any physiological and/or behavioral abnormality displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian SNORF25 receptor, the alleviation of such an abnormality identifying the compound as an antagonist.
This invention also provides an antagonist identified by the preceding method.
This invention further provides a composition, e.g. a pharmaceutical composition, comprising an antagonist according to this invention and a carrier, e.g. a pharmaceutically acceptable carrier.
This invention additionally provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF25 receptor which comprises administering to the subject an effective amount of the pharmaceutical composition according to this invention so as to thereby treat the abnormality.
In addition, this invention provides a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF25 receptor comprising administering a compound to a transgenic, nonhuman mammal according to this invention, and determining whether the compound alleviates any physiological and/or behavioral abnormality displayed by the transgenic, nonhuman mammal, the alleviation of such an abnormality identifying the compound as an agonist.
This invention further provides an agonist identified by the preceding method.
This invention still further provides a composition, e.g. a pharmaceutical composition, comprising an agonist according to this invention and a carrier, e.g. pharmaceutically acceptable carrier.
Moreover, this invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF25 receptor which comprises administering to the subject an effective amount of the pharmaceutical composition according to this invention so as to thereby treat the abnormality.
Yet further, this invention provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DNA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mammalian SNORF25 receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian SNORF25 receptor to create a unique band pattern specific to the DNA of subjects h suffering from the disorder; (f) repeating steps (a)-(e) with DNA obtained for diagnosis from subjects not yet suffering from the disorder; and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) with the band pattern from step (f) for subjects not yet suffering from the disorder so as to determine whether the patterns are the same or different and thereby diagnose predisposition to the disorder if the patterns are the same.
This invention also provides a method of preparing a purified mammalian SNORF25 receptor according to the invention which comprises: (a) culturing cells which express the mammalian SNORF25 receptor; (b) recovering the mammalian SNORF25 receptor from the cells; and (c) purifying the mammalian SNORF25 receptor so recovered.
This invention further provides a method of preparing the purified mammalian SNORF25 receptor according to the invention which comprises: (a) inserting a nucleic acid encoding the mammalian SNORF25 receptor into a suitable expression vector; (b) introducing the resulting vector into a suitable host cell; (c) placing the resulting host cell in suitable conditions permitting the production of the mammalian SNORF25 receptor; (d) recovering the mammalian SNORF25 receptor so produced; and optionally (e) isolating and/or purifying the mammalian SNORF25 receptor so recovered.
Furthermore, this invention provides a process for determining whether a chemical compound is a mammalian SNORF25 receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF25 receptor with the compound under conditions permitting the activation of the mammalian SNORF25 receptor, and detecting any increase in mammalian SNORF25 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF25 receptor agonist.
This invention also provides a process for determining whether a chemical compound is a mammalian SNORF25 receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian SNORF25 receptor with the compound in the presence of a known mammalian SNORF25 receptor agonist, under conditions permitting the activation of the mammalian SNORF25 receptor, and detecting any decrease in mammalian SNORF25 receptor activity, so as to thereby determine whether the compound is a mammalian SNORF25 receptor antagonist.
This invention still further provides a composition, for example a pharmaceutical composition, which comprises an amount of a mammalian SNORF25 receptor agonist determined by a process according to this invention effective to increase activity of a mammalian SNORF25 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
Also, this invention provides a composition, for example a pharmaceutical composition, which comprises an amount of a mammalian SNORF25 receptor antagonist determined by a process according to this invention effective to reduce activity of a mammalian SNORF25 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
This invention moreover provides a process for determining whether a chemical compound specifically binds to and activates a mammalian SNORF25 receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with the chemical compound under conditions suitable for activation of the mammalian SNORF25 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change, e.g. an increase, in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian SNORF25 receptor.
This invention still further provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian SNORF25 receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian SNORF25 receptor, wherein such cells do not normally express the mammalian SNORF25 receptor, with both the chemical compound and a second chemical compound known to activate the mammalian SNORF25 receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian SNORF25 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change, e.g. increase, in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian SNORF25 receptor.
In an embodiment of the invention, the second compound is a lipid-like molecule including, but not limited to, ATRA and phospholipids. Examples of phospholipids include, but are not limited to, PAF(C18), PAF(C16), lyso-PAF(C18) and lyso-PAF(C16).
Further, this invention provides a compound determined by a process according to the invention and a composition, for example, a pharmaceutical composition, which comprises an amount of a mammalian SNORF25 receptor agonist determined to be such by a process according to the invention, effective to increase activity of the mammalian SNORF25 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
This invention also provides a composition, for example, a pharmaceutical composition, which comprises an amount of a mammalian SNORF25 receptor antagonist determined to be such by a process according to the invention, effective to reduce activity of the mammalian SNORF25 receptor and a carrier, for example, a pharmaceutically acceptable carrier.
This invention yet further provides a method of screening a plurality of chemical compounds not known to activate a mammalian SNORF25 receptor to identify a compound which activates the mammalian SNORF25 receptor which comprises: (a)contacting cells transfected with and expressing the mammalian SNORF25 receptor with the plurality of compounds not known to activate the mammalian SNORF25 receptor, under conditions permitting activation of the mammalian SNORF25 receptor; (b) determining whether the activity of the mammalian SNORF25 receptor is increased in the presence of one or more the compounds; and if so (c) separately determining whether the activation of the mammalian SNORF25 receptor is increased by any compound included in the plurality of compounds, so as to thereby identify each compound which activates the mammalian SNORF25 receptor.
This invention provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian SNORF25 receptor to identify a compound which inhibits the activation of the mammalian SNORF25 receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian SNORF25 receptor with the plurality of compounds in the presence of a known mammalian SNORF25 receptor agonist, under conditions permitting activation of the mammalian SNORF25 receptor; (b) determining whether the extent or amount of activation of the mammalian SNORF25 receptor is reduced in the presence of one or more of the compounds, relative to the extent or amount of activation of the mammalian SNORF25 receptor in the absence of such one or more compounds; and if so (c) separately determining whether each such compound inhibits activation of the mammalian SNORF25 receptor for each compound included in the plurality of compounds, so as to thereby identify any compound included in such plurality of compounds which inhibits the activation of the mammalian SNORF25 receptor.
This invention also provides a composition, for example a pharmaceutical composition, comprising a compound identified by a method according to this invention in an amount effective to increase mammalian SNORF25 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
This invention still further provides a composition, for example, a pharmaceutical composition, comprising a compound identified by a method according to this invention in an amount effective to decrease mammalian SNORF25 receptor activity and a carrier, for example, a pharmaceutically acceptable carrier.
Furthermore, this invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian SNORF25 receptor which comprises administering to the subject a compound which is a mammalian SNORF25 receptor agonist in an amount effective to treat the abnormality.
This invention additionally provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian SNORF25 receptor which comprises administering to the subject a compound which is a mammalian SNORF25 receptor antagonist in an amount effective to treat the abnormality.
This invention also provides a process for making a composition of matter which specifically binds to a mammalian SNORF25 receptor which comprises identifying a chemical compound using a process in accordance with this invention and then synthesizing the chemical compound or a novel structural and functional analog or homolog thereof.
This invention further provides a process for preparing a composition, for example, a pharmaceutical composition which comprises admixing a carrier, for example, a pharmaceutically acceptable carrier, and a pharmaceutically effective amount of a chemical compound identified by a process in accordance with this invention or a novel structural and functional analog or homolog thereof.